We have previously demonstrated that intracellular single-chain antibodies sFv intrabodies can cause phenotypic knock-out of cell surface receptors. In this revised proposal, we provide preliminary data that relatively low affinity N-terminal directed human anti-CCR5 sFv intrabodies can inhibit the infection of Mphi-tropic strains of HIV-1. The theme of this revised proposal is to obtain new high affinity anti-CCR5 sFv intrabodies against different linear and conformational epitopes on CCR5 and evaluate their use as research tools to study gp120/CCR5 molecular interactions and as molecules for gene therapy of HIV-1 infection. Accordingly, we will determine through transient transfection experiments the level of expression, stability and the ability of the anti-CCR5 sFv intrabodies we have already constructed to specifically bind to nascent intracellular CCR5 and block protein transport to the cell surface and infection of single round HIV-1 CAT and GFP viruses that have been pseudotyped with Mphi-tropic envelope glycoproteins. We will determine if phenotypic knock-out of CCR5 in stably transfected PM1 cells will result in inhibition of infection with Mphi-tropic viruses and whether adaptation of HIV-1 to use CXCR4 occurs in the CCR5 knockout cell lines. We will also engineer two murine anti-CCR5 mAbs that are directed against linear epitopes on the extracellular N-terminal and 2nd extracellular loops, respectively, as sFv antibodies and intrabodies. Their binding affinities to the corresponding CCR5 polypeptides and to cells, their ability to neutralize HIV-1-infection of PBMCs and their intracellular expression as sFv intrabodies will be examined. We also plan to increase the affinity of our highest affinity N-terminal directed human anti-CCR5 sFv intrabody to at least 1 nM (70 fold increase) by sequentially randomizing amino acids in the VL and VH CDR3. We will also generate new anti-CCR5 sFvs from our non-immune human sFv library and create a new sFv phage display library from a immune mouse spleen that has been immunized with CCR5-L1.2 cells. Finally, to introduce the most potent anti-CCR5 sFv intrabodies into primary cells, we will use both MuLV retroviral vectors to transduce PBMCs and purified CD4+ mononuclear cells and HIV-1 vectors to transduce non-dividing CD4+ monocytes and Mphi from uninfected and HIV-1-infected individuals and determine of these transduced populations of cells can be protected from challenge with Mphi-tropic and NSI primary viruses in the former and autologous viruses in the latter. If successful, these studies will set the foundation for a new approach to the gene therapy of HIV-1 infection particularly if this approach can be applied in the future to the transduction of CD34+ bone marrow stem cells and/or to large scale replacement of transduced CD4+ PBMCs.